Design and synthesis of protein kinase C epsilon selective diacylglycerol lactones (DAG-lactones)

Article abstract of DOI:10.1016/j.ejmech.2014.11.025

DAG-lactones afford a synthetically accessible, high affinity platform for probing structure activity relationships at the C1 regulatory domain of protein kinase C (PKC). Given the central role of PKC isoforms in cellular signaling, along with their differential biological activities, a critical objective is the design of isoform selective ligands. Here, we report the synthesis of a series of DAG-lactones varying in their side chains, with a particular focus on linoleic acid derivatives. We evaluated their selectivity for PKC epsilon versus PKC alpha both under standard lipid conditions (100% phosphatidylserine, PS) as well as in the presence of a nuclear membrane mimetic lipid mixture (NML). We find that selectivity for PKC epsilon versus PKC alpha tended to be enhanced in the presence of the nuclear membrane mimetic lipid mixture and, for our lead compound, report a selectivity of 32-fold.

Full text of DOI:10.1016/j.ejmech.2014.11.025

Accepted Manuscript
Design and synthesis of protein kinase C epsilon selective diacylglycerol lactones
(DAG-Lactones)
Jihyae Ann , Suyoung Yoon , Jisoo Baek , Da Hye Kim , Nancy E. Lewin , Colin S.
Hill , Peter M. Blumberg , Jeewoo Lee
PII:
S0223-5234(14)01047-2
10.1016/j.ejmech.2014.11.025
EJMECH 7518
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 August 2014
Revised Date: 29 October 2014
Accepted Date: 11 November 2014
Please cite this article as: J. Ann, S. Yoon, J. Baek, D.H. Kim, N.E. Lewin, C.S. Hill, P.M. Blumberg, J.
Lee, Design and synthesis of protein kinase C epsilon selective diacylglycerol lactones (DAG-Lactones),
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.11.025.
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Graphical Abstract

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Design and Synthesis of Protein Kinase C Epsilon Se-
lective Diacylglycerol Lactones (DAG-Lactones)
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Jihyae Ann , Suyoung Yoon , Jisoo Baek , Da Hye Kim , Nancy E. Lewin , Colin S. Hill ,
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Peter M. Blumberg b, Jeewoo Lee a,*
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a
Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of
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Pharmacy, Seoul National University, Seoul 151-742, Korea
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Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer In-
stitute, NIH, Bethesda, MD 20892, USA
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* To whom correspondence should be addressed.
Phone: 82-2-880-7846
Fax: 82-2-888-0649
E-mail: jeewoo@snu.ac.kr
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Key Words: Protein Kinase C, Diacylglycerol Lactone, PKC-ε
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Abstract
DAG-lactones afford a synthetically accessible, high affinity platform for probing structure ac-
tivity relationships at the C1 regulatory domain of protein kinase C (PKC). Given the central role
of PKC isoforms in cellular signaling, along with their differential biological activities, a critical
objective is the design of isoform selective ligands. Here, we report the synthesis of a series of
DAG-lactones varying in their side chains, with a particular focus on linoleic acid derivatives.
We evaluated their selectivity for PKC epsilon versus PKC alpha both under standard lipid con-
ditions (100% phosphatidylserine, PS) as well as in the presence of a nuclear membrane mimetic
lipid mixture (NML). We find that selectivity for PKC epsilon versus PKC alpha tended to be
enhanced in the presence of the nuclear membrane mimetic lipid mixture and, for our lead com-
pound, report a selectivity of 32-fold.
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1. Introduction
Protein kinase Cs (PKCs) represent a family of serine/threonine kinases which are central
signaling elements downstream of the numerous cellular receptors that are coupled to phospho-
lipase C activation [1]. PKCs display complex regulation, with membrane phospholipids and di-
acylglycerol being critical regulators and elevated Ca2+ additionally being important for the clas-
sical PKCs. PKC isoforms divide into three groups; classical PKCs (cPKCs: PKCα, PKCβI,
PKCβII and PKCγ), novel PKCs (nPKCs: PKCδ, PKCε, PKCη and PKCθ) and atypical PKCs
(aPKCs: PKCξ, PKCι/λ), distinguished by their Ca2+ dependency and cofactors for activation
[2,3]. Additionally, individual isozymes vary in their patterns of tissue distribution, subcellular
localization, substrate specificity, and biological function [4,5]. For example, different isoforms
may be growth promoting or growth inhibitory, with stimulation of one isoform being function-
ally antagonistic of another [6]. Although the high level of conservation of the regulatory do-
mains of PKC isoforms poses a challenge for the development of isoform selective ligands, the
complexity of their regulation also provides promising opportunities [7].
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Among the PKC isozymes, particular attention has focused on protein kinase C epsilon
(PKC-ε), one of the calcium-independent but phorbol ester/ diacylglycerol dependent novel PKC
isoforms. Structurally, it contains an N-terminal regulatory domain and C-terminal kinase do-
main composed on four conserved (C1-C4) and five variable (V1-V5) regions. The regulatory
domain contains a pseudosubstrate domain which inhibits PKC activity before activation, a tan-
dem repeat of C1 domain zinc finger structures that bind diacylglycerol (DAG) and phorbol ester,
and a C2-like domain which contributes to membrane interaction [8,9]. Additionally, PKC-ε pos-
sesses a unique actin-binding site [10] between the first and second cysteine rich C1-domains
which promotes association with actin filaments in intact cells. Due to its specific tissue distribu-
tion and localization pattern, it has been reported that PKC-ε has critical roles in both the cardio-
vascular and central nervous system [11] such as cardioprotection [12], neurite outgrowth [13],
synapse formation, neurotransmitter release, synaptic loss prevention [14], and sensitization to
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pain through TRPV1 [15].
As an initial effort to design and develop PKC-ε selective ligands, our attention was
drawn to reports that saturated and unsaturated fatty acids may influence PKC-ε activity. Recent-
ly, Nishizaki and co-workers described that a linoleic acid derivative with cyclopropane rings
replacing the cis-double bonds, viz. 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic
acid (DCP-LA, Figure 2. Compound 5) acted as a selective and direct activator of PKC-ε [16]. In
biological studies, DCP-LA effectively prevented both deposition of amyloid plaques and loss of
synaptic connections [17].
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Previously, we have demonstrated that diacylglycerol lactones (DAG-lactones), which
are rigidified structures derived from the endogenous PKC ligand DAG, function as DAG ana-
logs to potently bind to the regulatory C1 domain of PKC and cause PKC activation [18]. For
this binding, both the acyl (R1) and α-alkylidene (R2) positions are critical elements in control-
ling biological activity and PKC isozyme selectivity [19]. Modeling reveals that there are two
different modes of binding, involving interaction between the C1 domain and either the sn-1 car-
bonyl or the sn-2 carbonyl. The alkyl chain which is not involved in hydrogen bonding to the
binding cleft of the C1 domain contributes to the binding energy through its interactions with the
C1 domain surface and the lipid bilayer.
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In this study, we have evaluated whether the incorporation of linoleic and DCP-LA deriv-
atives into the side chains of DAG-lactones would provide combined structures with enhanced
selectivity for PKCε. The newly designed linoleic acid derivatives, incorporating cyclo-saturated
(2, 5), linear-unsaturated (10, 12), linear-saturated (11), and branched alkyl chains (13-15), were
alternatively attached to the carbonyl group for acyl branching (R1) or connected to the lactone
via a methylene group for α-alkylidene branching (R2). Novel features of the study include both
the specific class of DAG-lactones themselves as well as their analysis, directed at comparing
selectivities for the novel PKCε isoform relative to that for the classical PKCα isoform.
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2. Result and discussion
2.1. Chemistry
As illustrated in Figure 2, three branched groups as well as linoleic acid and five different
linoleic acid derivatives in which the cis-double bonds were replaced by other groups were in-
troduced as R1 and R2 side chains. Commercially available linoleic acid (1), stearic acid (11), oc-
tadec-9-ynoic acid (12), and the branched compounds pivaloyl chloride (13) and isovaleralde-
hyde (15) were directly used for alkylation. Among the branched-substituents, 3-isobutyl-5-
methylhexanoyl chloride (14) [20] and 3-isobutyl-5-methylhexanal (16) [21] were prepared ac-
cording to previously published methods.
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Starting from linoleic acid 1, cyclo-saturated acid derivatives were synthesized. Di-epoxy
compound (DEP-LA, 2) was achieved by the conventional mCPBA epoxidation (Scheme 1).
Simmons-smith cyclopropanation [22] of methyl (9Z, 12Z)-octadeca-9,12-dienoate (3) followed
by hydrolysis of the ester gave the di-cyclopropane compound (DCP-LA, 5). The diyne core of
compound 10 was assembled by a Cadiot-Chodkiewicz coupling [23] between bromoalkyne 7
and a suitably acid functionalized terminal alkyne 9 (Scheme 2). As illustrated in scheme 3, the
aldehydes 20-22 were generated from the alcohols 17-19, which were derived from the corre-
sponding esters (3, 4) and acid (11), by treatment with the Dess-Martin periodinane.
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Scheme 1. Synthesis of cyclo-saturated alkyl acids.
Reagents and Conditions: (a) mCPBA, CH2Cl2, 0°C; (b) SOCl2, CH3OH, r.t.; (c) Et2Zn,CH2I2,CH2Cl2, -
5°C; (d) 1N LiOH, dioxane, 60°C.
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Scheme 2. Synthesis of the unsaturated-diyne derivative.
Reagents and Conditions: (a) NBS, AgNO3, Acetone, r.t.; (b) CrO3, H2SO4, H2O, Acetone,
-5°C; (c) CuCl, 30% n-BuNH2, NH2OHHCl, Et2O, r.t.
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Scheme 3. Synthesis of aldehydes for sn-2 alkylation
Reagents and Conditions: (a) LAH, Et2O, reflux; (b) (COCl)2, DMSO, Et3N, CH2Cl2, -78°C; (c) DMP,
CH2Cl2, 0°C.
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Depending on the specific substituents, different protecting groups were selected (Table
1). Specially, the TBDPS group was used for the synthesis of DEP-LA (90, 91) due to its sensi-
tivity to the deprotection conditions with BCl3 and CAN.
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Scheme 4. General scheme for alkylation.
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Reagents and Conditions: (a) i) LiHMDS, THF, R2CHO, -78°C. ii) MsCl, CH2Cl2, DBU; (b) BCl3,
CH2Cl2, -78°C or CAN, CH3CN/H2O; (c) TBAF, THF; (d) EDC, DMAP, CH2Cl2, r.t.; (e)
TEA, DMAP, CH2Cl2, r.t.; (f) BCl3, CH2Cl2, -78°C or CAN, CH3CN/H2O.
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The synthesis of DAG-lactone analogues started from the racemic lactone having p-
methoxyphenyl (PMP) and benzyl (Bn) (23) or tert-butyldiphenylsilyl (TBDPS) (24) as protect-
ing groups (Scheme 4). Different aldehydes (RCHO, 20-22) were reacted with the DAG-lactone
to form aldol intermediates and β-hydroxy-lactones were eliminated by aldol condensation to
give the corresponding olefins (25-45). After separation of geometric E/Z-isomers by silica col-
umn chromatography, parallel deprotection of 25-43 with BCl3 or ceric ammonium nitrate (CAN)
afforded 46-64 and di-deprotections of TBDPS protected compounds 44-45 with TBAF afforded
65-66. The compounds were individually converted to the corresponding DAG-lactones having
different R1-alkyl side chains by conventional coupling methods (90-91). Target compounds 86-
89, 92-106 were obtained from additional deprotection of 67-85 using BCl3 or CAN.
2.2. Biological Activity
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Diacylglycerol lactones 86-106 were prepared and investigated. For the biological studies,
we selected the classical PKC isoform PKC-α as the standard for comparison, since it has been
used as the standard PKC isoform against which all the DAG-lactones have routinely been char-
acterized in our previous studies. The interaction of the target DAG-lactones with PKC-α and
PKC-ε was assessed, as before, by measurement of the ability of the ligands to compete for bind-
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ing of [20-3H] phorbol 12, 13-dibutylate (PDBU) to recombinant PKC [24]. The IC50 values
were determined by least squares fit of the data points to the theoretical competition curve, and
the Ki values for inhibition of binding were calculated from the corresponding IC50 values (Table
2 and Table 3).
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Our initial objective was to examine the possible selectivity of the DAG-lactones for
PKC-ε under standard lipid conditions, viz. 100 ꢀg/ml phosphatidylserine. We determined the
binding affinities for PKC-α and PKC-ε and we calculated the Ki ratios for PKC-α/PKC-ε of all
compounds (Table 2, Table 3 and Table 4). Evaluation of the elements contributing to the relative
structure activity relations focused on geometry (E or Z), position of substitution (sn-1 or sn-2),
and lipophilicities of the compounds.
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The binding affinities of ligands for different PKC isozymes may show different depend-
ence on the lipid composition of the membranes with which the PKC is associated. We therefore
evaluated the ligand selectivity not only under our standard assay conditions, 100 ꢀg/ml of phos-
phatidylserine, which provides a highly anionic surface, but also using 100 ꢀg/ml of a lipid mix-
ture
[1-palmutoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphoethanolamine(POPE)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine
(POPS)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol(POPI)/ Cholesterol (61:21:4:7:6)], a
much less anionic lipid composition approximating that of the nuclear membrane.
Table 2 displays binding affinities of DAG-lactones under standard (100% phosphatidyl-
serine) conditions. Most of the compounds showed modestly stronger binding affinity for PKC-ε
than for PKC-α. The compounds with the strongest PKC-ε binding affinities were characterized
as having an E-geometry with a sn-1 carbonyl substituted with a branched alkyl chain or an un-
saturated alkyl chain (Table 2, compounds 95 and 104). The best, albeit limited PKCε selectivity
was seen for the sn-2 carbonyl substituted with a saturated alkyl chain (cyclo- 103, branched 104,
linear 105 and 106).
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The PKC-ε selectivity of the compounds was further investigated for the nuclear mem-
brane mimetic assay system (Table 3). The changes resulting from lipid composition were sub-
stantial, particularly for compound 104. The difference in lipid composition led to a reduction in
overall binding affinity and caused a different selectivity pattern to emerge. Compounds 91, 97,
99, and 104 showed over a 3-fold enhanced selectivity (Table 3). In particular, compound 104
retained its affinity to PKC-ε and showed the highest selectivity (32-fold) under the nuclear
membrane mimetic assay conditions (Table 3). The E-isomers having an sn-1 carbonyl substitut-
ed with an unsaturated alkyl chain showed good binding affinities and selectivity in this system.
A partial correlation with lipophilicity was observed among the compounds having over 5-fold
selectivity.
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Another important observation was the clear disparities in selectivity shown by the two
geometrical isomers (Table 4), with the E-isomer being favored particularly in the nuclear mem-
brane mimetic assay conditions.
3. Conclusion
In conclusion, different types of alkyl chains derived from linoleic acid were introduced
as substituents for the DAG-lactone and their effects on selectivity for PKC-ε relative to PKC-α
was explored. Because cells contain a variety of membranes with different compositions, we also
assessed the influence of membrane lipid contribution on the measured isozyme selectivity. Most
of the compounds showed at least modest PKC-ε selectivity with a number approaching 5-fold in
the presence of the nuclear lipid mixture. The most dramatic example of selectivity, 32-fold, was
not with any of the linoleic derivative substituted DAG-lactones, however, suggesting that the
incorporation of the linoleic acid motif into the DAG-lactone did not confer the hoped for selec-
tivity.
Structure activity relationships were assessed for selected compounds showing the great-
er extents of selectivity for PKC-ε. When 100% phosphatidylserine was used as the lipid, the E-
conformation was preferred for compounds in which the sn-2 carbonyl was substituted with a
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saturated alkyl chain (branched > linear > cyclo). In contrast, in the presence of the nuclear
membrane mimetic lipids, sn-1 substituted compounds with an E-conformation showed PKC-ε
selectivity regardless of whether the alkyl chains were saturated or unsaturated.
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The most selective compound under both lipid conditions was (E)-(2-(hydroxymethyl)-4-
(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl) methyl pivalate 104, which has the
sn-2 carbonyl substituted with a saturated-branched alkyl chain with an E-conformation. Com-
pound 104 showed a dramatic increase of selectivity (8-fold in the presence of phosphatidylser-
ine, 32-fold in the presence of the nuclear membrane mimetic lipid) in the nuclear membrane
conditions, reflecting that its binding affinity for PKC-α was significantly weaker under those
conditions while its affinity for PKC-ε was largely retained. In contrast, the other compounds
showed reduced binding affinities both for PKC-α and PKC-ε. This result suggests that the lipid
composition of the nuclear membrane enhanced the binding affinity to PKC-ε for 104, reflecting
enhanced interactions between the DAG-lactone side chain and the protein-membrane interface.
The diverse affinity patterns of compounds from this work provide guidance for future ap-
proaches. In particular, the high selectivity of 104 suggests that further exploration of this class
of DAG-lactones may be productive for obtaining PKC-ε specific compounds and understanding
of factors driving subcellular localization of PKC isoforms. Naturally, validation of the selectivi-
ty of the optimal PKC-ε selective ligand will ultimately require characterization relative to the
complete range of targets for DAG and phorbol esters, e.g. PKC family members, RasGRP, the
chimaerins, and include measures of activity in intact cells.
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4. Experimental
4.1. Chemistry
All chemical reagents were commercially available. Silica gel column chromatography was per-
formed on silica gel 60, 230-400 mesh, Merck. Nuclear magnetic resonance (1H-NMR) spectra
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were recorded on a JEOL JNM-LA 300 and a Bruker Avance 400 MHz FT-NMR spectrometer.
Chemical shifts are reported in ppm units with Me4Si as a reference standard. Mass spectra were
recorded on a VG Trio-2 GC-MS and a 6460 Triple Quad LC/MS. Elemental analyses were per-
formed with an EA 1110 Automatic Elemental Analyzer, CE Instruments.
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4.1.1. Preparation of alkyl side chains.
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4.1.1.1. 8-(3-((3-Pentyloxiran-2-yl)methyl)oxiran-2-yl)octanoic acid (2)
A solution of linoleic acid 1 (500 mg, 1.783 mmol) and meta-chloroperoxybenzoic acid (1.8 g,
10.698 mmol) in 10 ml of CH2Cl2 was reacted at 0°C for 3 h. The excess reagent was decom-
posed by addition of aqueous Na2CO3 and stirring of the mixture for 1 h, after which the product
was isolated by CH2Cl2 extraction. The organic layer was washed with saturated aqueous sodium
bicarbonate and saturated brine, dried with MgSO4, and evaporated. The crude product was puri-
fied by silica gel flash column chromatography (n-hexane/EtOAc, 4:1) and 2 (469 mg, 82%) was
obtained as a white solid.
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4.1.1.2. Methyl (9Z,12Z)-octadeca-9,12-dienoate (3)
Thionyl chloride (0.5ml, 6.239 mmol) was added dropwise to commercially available linoleic
acid 1 (700mg, 2.495 mmol) dissolved in MeOH at 0°C. The reaction was stirred at room tem-
perature and monitored by TLC. Upon completion, H2O was added and the reaction mixture was
concentrated in vacuo. The resulting solution was extracted with ethyl acetate, dried over MgSO4,
and concentrated in vacuo. The crude product was purified by silica gel flash column chromatog-
raphy (n-hexane/EtOAc, 10:1) and 3 (712 mg, 96%) was obtained as a brown oil.
4.1.1.3. Methyl 8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octanoate (5)
To a solution of linoleic acid methyl ester 3 (1 g, 3.395 mmol) in CH2Cl2, a 1.1 M n-hexane solu-
tion of diethyl zinc (7 ml, 40.751 mmol) was added under a N2 atmosphere with cooling with an
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ice-water bath (-5°C to 0°C) and was stirred for 1 h. Diiodomethane (7 ml, 81.502 equiv.) was
then added and the mixture was stirred at ambient temperature overnight. After removal of sol-
vent by evaporation, the product was extracted by ethyl acetate (EA) and purified by chromatog-
raphy on silica gel to give methyl 8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octanoate 4
(969 mg, 88%) as an oil. A mixture of this ester 4, 1N LiOH (6.6 mL), and dioxane was stirred at
60°C overnight. The product was purified by acid-base workup with 1N HCl and 1N NaOH and
5 (740mg, 80%) was obtained as an oil.
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4.1.1.4. Octadeca-9,11-diynoic acid (10)
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Copper (I) chloride (8.8 mg, 0.089 mmol.) was added to a 30% w/v n-BuNH2 aqueous solution
(10 ml) at room temperature, which resulted in the formation of a blue solution. A few crystals of
hydroxylamine hydrochloride was added to discharge the blue color. Dec-9-ynoic acid 9 (300mg,
1.78 mmol) was then added neat, and the mixture was stirred at 0°C for 10 min by which time
the formation of a bright yellow precipitate (probably the alkynyl cuprate) was observed. Bromo-
alkyne 6 (404 mg, 2.14 mmol) dissolved in diethyl ether was then added to the cooled reaction
mixture and the cooling bath was removed after 10-15 min. Occasional addition of a small
amount of hydroxylamine hydrochloride was necessary to keep the color of the reaction light
yellow. The reaction mixture was stirred at room temperature for 2 h during which more crystals
of hydroxylamine hydrochloride were added whenever the reaction mixture started to turn blue
or light green. The reaction was then quenched and adjusted to pH ~2 by adding 1N HCl and the
organic compounds were extracted with ethyl acetate 3 times. The combined layer was dried
over Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel flash col-
umn chromatography (n-hexane/EtOAc, 4:1) and 10 (423 mg, 86%) was obtained as a yellow oil.
4.1.1.4.1. 1-Bromohept-1-yne (7)
The terminal alkyne 6 (1ml, 6.350 mmol) was dissolved in acetone. N-bromosuccinimide (1.4 g,
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7.866 mmol) and AgNO3 (108 mg, 0.635 mmol) were added to the resulting solution in this or-
der, and the mixture was stirred at room temperature for 3 h, until complete consumption of the
starting material according to TLC. It was then poured into iced water. The aqueous layer was
extracted with pentane (3 times), and the combined organic extracts were dried over MgSO4, fil-
tered, and the solvent removed by evaporation under reduced pressure. The residue was purified
by silica gel flash column chromatography (n-hexane/EtOAc, 10:1) to yield bromo-alkyne 7
(1.07 g, 89%) was obtained as a colorless oil.
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4.1.1.4.2. Dec-9-ynoic acid (9)
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To a solution of 9-decyn-1-ol 8 (5 ml, 28.20 mmol) in acetone at -5°C was slowly added chromic
acid solution prepared from CrO3 (4.23 g, 42.30 mmol), water (4 ml), and conc. H2SO4 (3 ml) at
0°C. The mixture was then stirred for 2 h and left overnight at room temperature, after which it
was extracted with diethyl ether, washed with water and brine, dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by silica gel flash column chromatography (n-
hexane/EtOAc, 7:1) to yield dec-9-ynoic acid 9 (3.8 g, 81%) as clear oil.
4.1.2. Procedure for aldehyde preparation.
4.1.2.1. Alcohol preparation (17-19).
Starting material (2, 5, 8) was dissolved in ether. Lithium aluminum hydride (2 equiv.) was add-
ed at 0°C and it was refluxed during 30 min. H2O was added dropwise at 0°C; the solution was
extracted with ether and the combined extracts were dried over MgSO4 and concentrated in vac-
uo. Finally, purification by silica gel flash column chromatography (n-hexane/EtOAc, 10:1) by
column chromatography yielded the alcohols (17-19).
4.1.2.2. Aldehyde preparation (20-22).
Method A. A solution of oxalyl chloride (1.5 equiv.) in distilled CH2Cl2 was cooled to -78°C,
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and DMSO (3 equiv.) was carefully added under a N2 atmosphere. After stirring for 15 min, a
solution of alcohol (17, 18) in CH2Cl2 was added followed by Et3N (6 equiv.). The cooling bath
was removed, and the reaction mixture was allowed to warm to room temperature and stirred for
2.5 h. The solvent was removed under reduced pressure, and the residue was extracted with ethyl
acetate. The extract was washed with saturated aqueous Na2CO3 solution and brine, and was then
dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the crude
product was purified by silica gel flash column chromatography (n-hexane/EtOAc, 10:1) and the
aldehyde (20, 21) was obtained as 75-80%.
3
4
5
6
7
8
9
Method B. To a solution of octadecan-1-ol 19 (560mg, 1equiv.) in CH2Cl2 was added Dess-
Martin periodinane (1.3g, 1.5 equiv.) at room temperature and the resulting solution was stirred
for 2 h at room temperature. The reaction mixture was quenched with a saturated solution of
Na2SO3 and NaHCO3. The reaction mixture was passed through a pad of Celite, the organic
compound was extracted three times with CH2Cl2. The combined extracts were washed with wa-
ter, dried over anhydrous MgSO4, and filtrated. The filtrate was concentrated under vacuum, and
purified by silica gel flash column chromatography (n-hexane/EtOAc, 10:1) by column chroma-
tography to afford the stearaldehyde 22 (456mg, 82%).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4.1.3. General procedure for Alkylation
A stirred solution of lactone 23 (or 24) (1 equiv.) in anhydrous THF (5 mL/mmol) was cooled to
-78°C under nitrogen and treated dropwise with LiHMDS (3 equiv., 2 M solution in THF). The
mixture was stirred at -78°C for 30-60 min and lithium enolate formation was detected by TLC
(n-hexane/EtOAc, 4:1). A solution of the corresponding aldehyde (1.4 to 4 equiv.) in anhydrous
THF (1 mL/mmol) was added dropwise to the enolate at the same temperature and stirring at -
78°C was continued for 1.5-2 h. The reaction was warmed to room temperature and quenched by
the slow addition of a saturated aqueous solution of ammonium chloride. The aqueous layer was
extracted three times with Et2O, and the combined organic extracts were washed three times with
14

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1
2
water, twice with brine, dried over MgSO4, and filtered. The filtrate was concentrated under vac-
uum to afford the crude alkylation reaction product, which was partially purified by silica gel
flash column chromatography after eluting with the appropriate solvent. The obtained mixture of
β-hydroxy-lactone diastereomers were typically used directly in the following step without fur-
ther purification.
3
4
5
6
7
4.1.4. General procedure for Mesylation-Olefination
8
A solution of the alkylation product in CH2Cl2 at 0°C was treated with methanesulfonyl chloride
(2 equiv.) and triethylamine (4 equiv.). The mixture was stirred at 0°C for 30 min and then for 2
h at room temperature. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 5 equiv.) was added at 0°C,
and the resulting solution was stirred overnight at ambient temperature. The reaction mixture
was concentrated to a brown syrup and the residue was extracted with EtOAc followed by 1N
HCl. The combined organic extracts were washed three times with water, twice with brine, dried
over MgSO4, and filtered. The combined organics were purified by silica gel flash column chro-
matography after eluting with the appropriate solvent.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4.1.5. General procedure for deprotection
4.1.5.1. PMP-deprotection
A stirred solution of the olefination product (1 equiv.) in acetonitrile/water (4/1) was treated with
CAN (3 equiv.) while under nitrogen at 0°C, and the reaction was monitored by TLC. After 30
min, the reaction was quenched by addition of aqueous NaHCO3 solution and the resulting solu-
tion was extracted with CH2Cl2, dried over MgSO4, and concentrated in vacuo. It was purified by
silica gel flash column chromatography (n-hexane/EtOAc, 2:1) and the deprotected compound
was obtained.
4.1.5.2. Benzyl Deprotection
15

ACCEPTED MANUSCRIPT
1
2
BCl3 (3 equiv.) was added slowly to a stirred solution of the olefination product (1 equiv.) in
CH2Cl2 at -78°C. The reaction was monitored by TLC and quenched by addition of MeOH at
0°C. The reaction mixture was concentration in vacuo and purification by silica gel flash column
chromatography (n-hexane/EtOAc, 2:1) gave the desired deprotected compounds.
3
4
5
6
4.1.5.3. TBDPS Deprotection
7
TBAF (2 equiv.) was added slowly to a stirred solution of the olefination product (1 equiv.) in
THF at 0°C and stirring continued as the temperature was allowed to rise to room temperature.
The reaction was monitored by TLC and the solution was concentrated in vacuo upon completion
without further work-up. Purification by silica gel flash column chromatography (n-
hexane/EtOAc, 1:1) gave the desired de-protected diol-lactone.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4.1.6. General procedure for Acylation
A solution of mono-deprotected compound (1 equiv.) in CH2Cl2 was treated with Et3N (3 equiv.)
and dimethylaminopyridine (DMAP, 2.5 equiv.) and reacted with the corresponding acid chloride
(RCOCl, 1.2-1.5 equiv.). The reaction was stirred at room temperature and monitored by TLC.
Upon completion, the reaction was concentrated in vacuo and purified by silica gel flash column
chromatography give the desired compound.
4.1.6.1. Monoacylation (di-epoxy compounds, DEP-LA)
A solution of 65 (or 66) (1 equiv.) in CH2Cl2 was treated with EDC (1.1 equiv.) and dimethyla-
minopyridine (DMAP, 0.3 equiv.) and reacted with 8-(3-((3-pentyloxiran-2-yl)methyl)oxiran-2-
yl)octanoic acid (1.5 equiv.). The reaction was stirred at room temperature and monitored by
TLC. Upon completion, the reaction was terminated by adding H2O. The organic layer was ex-
tracted with CH2Cl2, dried over MgSO4, concentrated in vacuo and purified by silica gel flash
column chromatography (n-hexane/EtOAc, 4:1) to give the mono-substituted desired compound
16

ACCEPTED MANUSCRIPT
1
2
90-91.
3
4.1.7. ((Z)-2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl-
(9Z,12Z)-octadeca-9,12-dienoate (86)
4
5
Yield 63%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.24 (t, J = 7.76 Hz, 1H, >C=CHCH2CH(CH3)2),
5.39–5.30 (m, 4H, -CH=CHCH2CH=CH-), 4.26 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH),
4.14 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 3.58-3.69 (dq, J = 12.00, 7.20, 6.56 Hz, 2H,
HOCH2-), 2.88 (AB d, J = 16.44 Hz, 1H, H-3a), 2.75 (t, J = 6.24 Hz, 2H, -CH=CHCH2CH=CH-),
2.72 ( AB d, J = 16.44 Hz, 1H, H-3b), 2.60 (t, J = 7.44 Hz, 2H, >C=CHCH2CH(CH3)2), 2.31 (t, J
= 7.56 Hz, 2H, Alkyl-CH2(O)COCH2), 2.04 (q, J = 6.88 Hz, 4H, -CH2CH=CHCH2CH=CHCH2-),
1.96 (m, 1H, HOCH2-), 1.71 (septet, J = 6.74 Hz, 1H, >C=CHCH2CH(CH3)2), 1.59 (m, 2H,
Alkyl-CH2CH2(O)COCH2), 1.28 (m, 14H, CH3(CH2)3CH2CH=CHCH2CH=CHCH2(CH2)4CH2-),
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
0.92 (d, J = 6.68 Hz, 6H,
>C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz, 3H,
CH3(CH2)3CH2CH=CH-); MS (FAB) m/z 477 (M+H)
4.1.8. ((E)-2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
(9Z,12Z)-octadeca-9,12-dienoate (87)
Yield 76%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.78 (t, J = 7.76 Hz, 1H, >C=CHCH2CH(CH3)2),
5.39–5.30 (m, 4H, -CH=CHCH2CH=CH-), 4.26 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH),
4.14 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 3.58-3.69 (dq, J = 12.00, 7.20, 6.56 Hz, 2H,
HOCH2-), 2.73-2.81 (m, 3H, H-3a, -CH=CHCH2CH=CH-), 2.62 ( AB d, J = 17.04 Hz, 1H, H-3b),
2.31 (t, J = 7.56 Hz, 2H, Alkyl-CH2(O)COCH2), 2.04 (q, J = 6.88 Hz, 4H, -
CH2CH=CHCH2CH=CHCH2-), 1.96 (m, 1H, HOCH2-), 1.71 (septet, J = 6.74 Hz, 1H,
>C=CHCH2CH(CH3)2), 1.59 (m, 2H, Alkyl-CH2CH2(O)COCH2), 1.28 (m, 14H,
CH3(CH2)3CH2CH=CHCH2CH=CHCH2(CH2)4CH2-), 0.92 (d,
J
=
6.68 Hz, 6H,
>C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz, 3H, CH3(CH2)3CH2CH=CH-)-); MS (FAB) m/z 477
17

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1
2
(M+H)
3
4.1.9. (Z)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl 8-
(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octanoate (88)
4
5
Yield 71%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.24 (t, J = 7.76 Hz, 1H, >C=CHCH2CH(CH3)2),
4.26 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 4.14 (AB d, J = 11.8 Hz, 1H, Alkyl-
(O)COCHH), 3.58-3.69 (dq, J = 12.1, 7.20, 6.56 Hz, 2H, HOCH2), 2.88 (AB d, J = 16.52 Hz, 1H,
H-3a), 2.69 ( AB q, J = 16.44 Hz, 1H, H-3b), 2.60 (t, J = 7.44 Hz, 2H, >C=CHCH2CH(CH3)2),
2.31 (t, J = 7.60 Hz, 2H, Alkyl-CH2(O)COCH2), 2.04 (t, J = 4.00 Hz, 1H, HOCH2), 1.69 (septet,
J = 6.72 Hz, 1H, >C=CHCH2CH(CH3)2), 1.59 (m, 2H, Alkyl-CH2CH2(O)COCH2,), 1.37 (bs, 6H,
Alkyl), 1.28 (bs, 12H, Alkyl), 1.10 (m, 2H, Alkyl), 0.92 (d, 6H, J = 6.60 Hz,
>C=CHCH2CH(CH3)2), 0.87 (t, J = 6.44 Hz, 3H, CH3-Alkyl-(O)COCH2), 0.75 (m, 2H, -
CHCH2CH-), 0.66 (m, 2H, -CHCH2CH-), 0.57 (m, 2H, -CHCH2CH-), -0.30 (m, 2H, -
CHCH2CH-); MS (FAB) m/z 505 (M+H)
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4.1.10. (E)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octanoate (89)
Yield 73%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.78 (t, J = 7.76 Hz, 1H, >C=CHCH2CH(CH3)2),
4.26 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 4.14 (AB d, J = 11.8 Hz, 1H, Alkyl-
(O)COCHH), 3.58-3.69 (dq, J = 12.1, 7.20, 6.56 Hz, 2H, HOCH2), 2.88 (AB d, J = 16.52 Hz, 1H,
H-3a), 2.69 ( AB q, J = 16.44 Hz, 1H, H-3b), 2.31 (t, J = 7.60 Hz, 2H, Alkyl-CH2(O)COCH2),
2.04 (m, 3H, HOCH2, >C=CHCH2CH(CH3)2),), 1.69 (septet, J = 6.72 Hz, 1H,
>C=CHCH2CH(CH3)2), 1.59 (m, 2H, Alkyl-CH2CH2(O)COCH2,), 1.37 (bs, 6H, Alkyl), 1.28 (bs,
12H, Alkyl), 1.10 (m, 2H, Alkyl), 0.92 (d, 6H, J = 6.60 Hz, >C=CHCH2CH(CH3)2), 0.87 (t, J =
6.44 Hz, 3H, CH3-Alkyl-(O)COCH2), 0.75 (m, 2H, -CHCH2CH-), 0.66 (m, 2H, -CHCH2CH-),
0.57 (m, 2H, -CHCH2CH-), -0.30 (m, 2H, -CHCH2CH-); MS (FAB) m/z 505 (M+H)
18

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1
2
3
4.1.11. (Z)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
8-(3-((3-pentyloxiran-2-yl)methyl)oxiran-2-yl)octanoate (90)
4
Yield 35%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.24 (t, J = 6.24 Hz, 1H, >C=CHCH2CH(CH3)2),
4.26 (AB d, J = 9.56 Hz, 1H, Alkyl-(O)COCHH), 4.14 (AB d, J = 9.56 Hz, 1H, Alkyl-
(O)COCHH), 3.59-3.68 (m, 2H, HOCH2-), 3.09 (bs, 2H, -CH(O)CH-), 2.95 (bs, 2H, -CH(O)CH-
), 2.78 (AB d, J = 13.00 Hz, 1H, H-3a), 2.68 (AB d, J = 13.00 Hz, 1H, H-3b), 2.60 (t, J = 5.72 Hz,
2H, >C=CHCH2CH(CH3)2), 2.31 (t, J = 5.96 Hz, 2H, Alkyl-CH2(O)COCH2-), 1.81 (m, 1H,
>C=CHCH2CH(CH3)2), 1.70 (m, 3H, -CH(O)CHCH2CH(O)CH-), 1.31-1.40 (m, 20H, Alkyl),
0.92 (d, J = 5.28 Hz, 6H, >C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz, 3H, CH3-Alkyl-); MS
(FAB) m/z 509 (M+H)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4.1.12. (E)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
8-(3-((3-pentyloxiran-2-yl)methyl)oxiran-2-yl)octanoate (91)
Yield 32%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.24 (m, 1H, >C=CHCH2CH(CH3)2), 4.26 (AB d,
J = 9.52 Hz, 1H, Alkyl-(O)COCHH), 4.14 (AB d, J = 9.52 Hz, 1H, Alkyl-(O)COCHH), 3.60-
3.71 (dq, J = 9.66, 5.64, 5.24 Hz, 2H, HOCH2-), 3.09 (bs, 2H, -CH(O)CH-), 2.95 (bs, 2H, -
CH(O)CH-), 2.78 (AB d, J = 14.60 Hz, 1H, H-3a), 2.62 (AB d, J = 14.60 Hz, 1H, H-3b), 2.31 (t,
J = 6.00 Hz, 2H, Alkyl-CH2(O)COCH2-), 2.11 (m, 1H, HOCH2-), 2.06 (m, 2H,
>C=CHCH2CH(CH3)2), 1.81 (m, 1H, >C=CHCH2CH(CH3)2), 1.70 (m, 3H,
-
CH(O)CHCH2CH(O)CH-), 1.44-1.49 (m, 8H, Alkyl), 1.22-1.32 (m, 12H, Alkyl), 0.92 (d, J =
5.28 Hz, 6H, >C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz, 3H, CH3-Alkyl-); MS (FAB) m/z 509
(M+H)
4.1.13. (Z)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
stearate (92)
19

ACCEPTED MANUSCRIPT
1
2
Yield 60%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.24 (t, J = 7.72 Hz, 1H, >C=CHCH2CH(CH3)2),
4.26 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 4.14 (AB d, J = 11.8 Hz, 1H, Alkyl-
(O)COCHH), 3.64 (m, 2H, HOCH2-), 2.90 (AB d, J = 16.34 Hz, 1H, H-3a), 2.69 (AB d, J =
16.34 Hz, 1H, H-3b), 2.60 (t, J = 7.48 Hz, 2H, >C=CHCH2CH(CH3)2), 2.30 (t, J = 7.44 Hz, 2H,
Alkyl-CH2(O)COCH2-), 2.04 (m, 1H, HOCH2-), 1.71 (m, 1H, >C=CHCH2CH(CH3)2), 1.58 (m,
2H), 1.23 (m, 28H, Alkyl), 0.92 (d, J = 6.64 Hz, 6H, >C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz,
3H, CH3-Alkyl-); MS (FAB) m/z 481 (M+H)
3
4
5
6
7
8
9
4.1.14. (E)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
stearate (93)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Yield 60%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.78 (m, 1H, >C=CHCH2CH(CH3)2), 4.26 (AB d,
J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 4.15 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 3.66 (q,
J = 11.04 Hz, 2H, HOCH2-), 2.79 (AB d, J = 16.00Hz, 1H, H-3a), 2.63 (AB d, J = 16.00 Hz, 1H,
H-3b), 2.30 (t, J = 7.64 Hz, 2H, Alkyl-CH2(O)COCH2-), 2.04 (m, 3H, HOCH2-,
>C=CHCH2CH(CH3)2),), 1.80 (septet, J = 6.74 Hz, 1H, >C=CHCH2CH(CH3)2), 1.23 (m, 32H,
Alkyl), 0.92 (d, J = 6.64 Hz, 6H, >C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz, 3H, CH3-Alkyl-);
MS (FAB) m/z 481 (M+H)
4.1.15. (Z)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
3-isobutyl-5-methylhexanoate (94)
Yield 59%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.26 (tt, J = 7.28, 2.3 Hz, 1H,
>C=CHCH2CH(CH3)2), 4.20 (AB q, J = 11.9 Hz, 2H, Alkyl-(O)COCH2), 3.66 (AB q, J = 12.2
Hz, 2H, HOCH2-), 2.90 (AB d, J = 16.4Hz, 1H, H-3a), 2.72 (AB d, J = 16.4 Hz, 1H, H-3b), 2.62
(m, 2H, >C=CHCH2CH(CH3)2), 2.24 (m, 2H, ((CH3)2CHCH2)2CHCH2(O)COCH2-), 1.80 (m, 1H,
>C=CHCH2CH(CH3)2), 1.68-1.78 (m, 1H, ((CH3)2CHCH2)2CHCH2(O)COCH2-), 1.58-1.62 (m,
2H, ((CH3)2CHCH2)2CHCH2(O)COCH2-), 1.03 (m, 4H, ((CH3)2CHCH2)2CHCH2(O)COCH2-),
20

ACCEPTED MANUSCRIPT
1
2
0.96
(d,
J
=
6.70
Hz,
6H,
>C=CHCH2CH(CH3)2),
0.86
(m,
12H,
((CH3)2CHCH2)2CHCH2(O)COCH2-); MS (FAB) m/z 383 (M+H)
3
4
4.1.16. (E)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
3-isobutyl-5-methylhexanoate (95)
5
6
Yield 62%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.80 (tt, J = 7.70, 2.3 Hz, 1H,
>C=CHCH2CH(CH3)2), 4.31 (AB d, J = 3.12 Hz, 1H, Alkyl-(O)COCHH), 4.17 (AB d, J = 2.76
Hz, 1H, Alkyl-(O)COCHH), 3.68 (m, 2H, HOCH2-), 2.83 (AB d, J = 17.01 Hz, 1H, H-3a), 2.65
(AB d, J = 17.01 Hz, 1H, H-3b), 2.22-2.24 (m, 2H, ((CH3)2CHCH2)2CHCH2(O)COCH2-), 2.04
(m, 3H, HOCH2-, >C=CHCH2CH(CH3)2), 1.82 (m, 1H, >C=CHCH2CH(CH3)2), 1.61 (m, 1H,
((CH3)2CHCH2)2CHCH2(O)COCH2-), 1.58 (m, 2H, ((CH3)2CHCH2)2CHCH2(O)COCH2-), 1.03
(m, 4H, ((CH3)2CHCH2)2CHCH2(O)COCH2-), 0.96 (d, J = 6.70 Hz, 6H, >C=CHCH2CH(CH3)2),
0.86 (m, 12H, ((CH3)2CHCH2)2CHCH2(O)COCH2-); MS (FAB) m/z 383 (M+H)
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4.1.17. (Z)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
octadec-9-ynoate (96)
Yield 80%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.24 (m, 1H, >C=CHCH2CH(CH3)2), 4.26 (AB d,
J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 4.14 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 3.61 (q,
J = 12.2 Hz, 2H, HOCH2-), 2.88 (AB d, J = 17.32, 2.2 Hz, 1H, H-3a), 2.63 ( AB d, J = 17.32, 2.2
Hz, 1H, H-3b), 2.60 (tt, J = 7.24, 2.0 Hz, 2H, >C=CHCH2CH(CH3)2), 2.30 (t, J = 7.52 Hz, 2H,
Alkyl-CH2(O)COCH2-), 2.12 (t, J = 6.24 Hz, 4H, -CH2C≡CCH2-), 1.71 (septet, J = 6.72 Hz, 1H,
>C=CHCH2CH(CH3)2), 1.59 (m, 7H), 1.44 (m, 5H), 1.25 (m, 11H), 0.92 (d, J = 6.68 Hz, 6H,
>C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz, 3H, CH3-Alkyl); MS (FAB) m/z 477 (M+H)
4.1.18. (E)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
octadec-9-ynoate (97)
21

ACCEPTED MANUSCRIPT
1
2
Yield 77%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.74 (m, 1H, >C=CHCH2CH(CH3)2), 4.26 (AB d,
J = 11.88 Hz, 1H, Alkyl-(O)COCHH), 4.17 (AB d, J = 11.88 Hz, 1H, Alkyl-(O)COCHH), 3.66
(m, 2H, HOCH2-), 2.81 (AB d, J = 17.19 Hz, 1H, H-3a), 2.64 ( AB d, J = 17.19 Hz, 1H, H-3b),
2.32 (t, J = 7.68 Hz, 2H, Alkyl-CH2(O)COCH2-), 2.13 (t, J = 6.24 Hz, 4H, -CH2C≡CCH2-), 2.07
(m, 2H, >C=CHCH2CH(CH3)2), 1.71 (septet, J = 6.72 Hz, 1H, >C=CHCH2CH(CH3)2), 1.60 (m,
7H), 1.27-1.47 (m, 21H), 0.95 (d, J = 6.60 Hz, 6H, >C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz,
3H, CH3-Alkyl); MS (FAB) m/z 477 (M+H)
3
4
5
6
7
8
9
4.1.19. (Z)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
octadeca-9,11-diynoate (98)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Yield 74%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.25 (tt, J = 7.82, 7.78 Hz, 1H,
>C=CHCH2CH(CH3)2), 4.25 (AB d, J = 12.00 Hz, 1H, Alkyl-(O)COCHH), 4.14 (AB d, J =
12.00 Hz, 1H, Alkyl-(O)COCHH), 3.65 (dq, J = 7.2, 6.52 Hz, 2H, HOCH2-), 2.88 (AB d, J =
16.52 Hz, 1H, H-3a), 2.70 ( AB d, J = 16.52 Hz, 1H, H-3b), 2.60 (tt, J = 6.84, 2.04, 2.00 Hz, 2H,
>C=CHCH2CH(CH3)2), 2.30 (t, J = 7.48 Hz, 2H, Alkyl-CH2(O)COCH2-), 2.22 (t, J = 6.80 Hz,
4H, CH3(CH2)4CH2-C≡C-C≡C-CH2-), 2.06 (m, 1H, HOCH2-), 1.71 (septet, J = 6.72 Hz, 1H,
>C=CHCH2CH(CH3)2), 1.59 (m, 2H, Alkyl), 1.49 (m, 4H), 1.33-1.39 (m, 3H), 1.22-1.29 (m, 3H,
Alkyl), 0.93 (d, J = 6.68 Hz, 6H, >C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz, 3H, CH3-Alkyl);
MS (FAB) m/z 473 (M+H)
4.1.20. (E)-(2-(Hydroxymethyl)-4-(3-methylbutylidene)-5-oxotetrahydrofuran-2-yl)methyl
octadeca-9,11-diynoate (99)
Yield 74%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.78 (tt, J = 7.80, 7.68 Hz, 1H,
>C=CHCH2CH(CH3)2), 4.26 (AB d, J = 11.84 Hz, 1H, Alkyl-(O)COCHH), 4.15 (AB d, J =
11.84 Hz, 1H, Alkyl-(O)COCHH), 3.66 (dq, J = 7.00, 6.44 Hz, 2H, HOCH2-), 2.79 (AB d, J =
16.81 Hz, 1H, H-3a), 2.62 ( AB d, J = 16.81 Hz, 1H, H-3b), 2.30 (t, J = 7.34 Hz, 2H, Alkyl-
22

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2
CH2(O)COCH2-), 2.22 (t, J = 6.92 Hz, 4H, CH3(CH2)4CH2-C≡C-C≡C-CH2-), 2.06 (m, 3H,
HOCH2-, >C=CHCH2CH(CH3)2), 1.80 (septet, J = 6.64 Hz, 1H, >C=CHCH2CH(CH3)2), 1.49
(m, 4H, Alkyl), 1.33-1.39 (m, 3H), 1.22-1.29 (m, 9H, Alkyl), 0.93 (d, J = 6.64 Hz, 6H,
>C=CHCH2CH(CH3)2), 0.86 (t, J = 6.08 Hz, 3H, CH3-Alkyl); MS (FAB) m/z 473 (M+H)
3
4
5
6
4.1.21. ((Z)-2-(Hydroxymethyl)-4-((9Z,12Z)-octadeca-9,12-dien-1-ylidene)-5 oxotetrahydro-
furan-2-yl)methyl pivalate (100)
7
8
Yield 54%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.22 (t, J = 7.76 Hz, 1H, >C=CH(CH2)7-), 5.32 (m,
4H, -CH=CHCH2CH=CHCH2-), 4.28 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 4.12 (AB d, J
= 11.8 Hz, 1H, Alkyl-(O)COCHH), 3.58-3.69 (dq, J = 12.1, 7.04, 6.64 Hz, 2H, HOCH2), 2.80
(AB q, J = 14.64 Hz, 1H, H-3a), 2.61-2.76 ( m, 4H, H-3b,>C=CHCH2-, HOCH2-), 2.02 (m, 4H, -
CH2CH=CHCH2CH=CHCH2-), 1.72 (m, 2H, -CH2CH=CHCH2CH=CHCH2-), 1.28 (bs, 16H,
>C=CH(CH2)5CH2-, -(CH2)3CH3), 1.17 (s, 9H, (CH3) 3(O)COCH2), 0.85 (m, 3H, Alkyl-CH3);
MS (FAB) m/z 477 (M+H)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4.1.22. ((E)-2-(Hydroxymethyl)-4-((9Z,12Z)-octadeca-9,12-dien-1-ylidene)-5 oxotetrahydro-
furan-2-yl)methyl pivalate (101)
1
Yield 50%, Oil; H NMR (CDCl3, 400MHz) δ 6.75 (m, 1H, >C=CH(CH2)7-), 5.32 (m, 4H, -
CH=CHCH2CH=CHCH2-), 4.28 (AB d, J = 11.8 Hz, 1H, Alkyl-(O)COCHH), 4.12 (AB d, J =
11.8 Hz, 1H, Alkyl-(O)COCHH), 3.58-3.69 (dq, J = 12.1, 7.04, 6.64 Hz, 2H, HOCH2-), 2.80
(AB d, J = 17.08 Hz, 1H, H-3a), 2.63 (AB d, J = 17.08 Hz, 1H, H-3b), 2.14 (m, 2H, >C=CHCH2-
), 2.02 (m, 4H, -CH2CH=CHCH2CH=CHCH2-), 1.72 (m, 2H, -CH2CH=CHCH2CH=CHCH2-),
1.28 (bs, 16H, >C=CH(CH2)5CH2-, -(CH2)3CH3), 1.17 (s, 9H, (CH3) 3(O)COCH2), 0.85 (m, 3H,
Alkyl-CH3); MS (FAB) m/z 477 (M+H)
4.1.23. (Z)-(2-(Hydroxymethyl)-5-oxo-4-(8-(2-((2-
23

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1
2
pentylcyclopropyl)methyl)cyclopropyl)octylidene)tetrahydrofuran-2-yl)methylpivalate (102)
1
Yield 55%, Oil; H NMR (CDCl3, 400MHz) δ 6.22 (t, J = 7.36 Hz, 1H, >C=CH(CH2)7-), 4.25
3
(AB d, J = 12.00 Hz, 1H, (CH3)3(O)COCHH-), 4.11 (AB d, J = 12.00 Hz, 1H,
(CH3)3(O)COCHH-), 3.63 (dq, J = 18.96, 7.24, 6.60 Hz, 2H, HO-CH2), 2.88 (AB d, J = 15.54
Hz, 1H, H-3a), 2.67 (m, 3H, H-3b, >C=CHCH2(CH2)6-), 2.04 (t, J = 7.2 Hz, 1H, HO-CH2-), 1.11-
1.46 (m, 30H, Alkyl), 0.82 (m, 3H, -Alkyl-CH3); MS (FAB) m/z 505 (M+H)
4
5
6
7
8
4.1.24. (E)-(2-(Hydroxymethyl)-5-oxo-4-(8-(2-((2-
9
pentylcyclopropyl)methyl)cyclopropyl)octylidene)tetrahydrofuran-2-yl)methylpivalate (103)
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Yield 60%, Oil; H NMR (CDCl3, 400MHz) δ 6.75 (t, J = 7.2 Hz, 1H, >C=CH(CH2)7-), 4.28
(AB d, J = 12.00 Hz, 1H, (CH3)3(O)COCHH-), 4.13 (AB d, J = 12.00 Hz, 1H,
(CH3)3(O)COCHH-), 3.59-3.72 (dq, J = 14.52, 6.96, 6.48 Hz, 2H, HO-CH2-), 2.80 (AB d, J =
17.24 Hz, 1H, H-3a), 2.64 (AB-d, J = 17.24 Hz, 1H, H-3b), 2.15 (m, 2H, >C=CHCH2(CH2)6-),
2.05 (t, J = 6.84 Hz, HO-CH2), 1.10-1.52 (m, 22H, Alkyl), 1.05 (s, 9H, (CH3)3(O)COCH2-), 0.87
(m, 3H, -Alkyl-CH3), 0.64-0.69 (m, 2H) , 0.56-0.61 (m, 1H); MS (FAB) m/z 505 (M+H)
4.1.25. (E)-(2-(Hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-
yl)methyl pivalate (104)
1
Yield 59%, Oil; H NMR (CDCl3, 400MHz) δ 6.78 (m, 1H, >C=CHCH2CH(CH2(CH3)2)2), 4.29
(AB d, J = 11.8 Hz, 1H, (CH3)3(O)COCHH-), 4.11 (AB d, J = 11.8 Hz, 1H, (CH3)3(O)COCHH-),
3.59-3.72 (dq, J = 13.2, 7.16, 6.6 Hz, 2H, HO-CH2), 2.79 (AB d, J = 17.1 Hz, H-3a), 2.62 (AB d,
J = 17.1 Hz, 1H, H-3b), 2.11 (t, J = 6.04 Hz, 2H, >C=CHCH2CH(CH2(CH3)2)2), 2.03 (t, J = 6.88
Hz, 1H, HO-CH2), 1.69 (pentet, J = 6.56 Hz, 1H,>C=CHCH2CH(CH2(CH3)2)2), 1.06 (septet, J =
6.72 Hz, 2H, >C=CHCH2CH(CH2CH(CH3)2)2), 1.17 (s, 9H, (CH3)3(O)COCH2-), 1.04-1.10 (m,
4H,
>C=CHCH2CH(CH2CH(CH3)2)2),
0.85
(d,
J
=
6.44
Hz,
12H,
>C=CHCH2CH(CH2CH(CH3)2)2); MS (FAB) m/z 383 (M+H)
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2
4.1.26. (Z)-(2-(Hydroxymethyl)-4-octadecylidene-5-oxotetrahydrofuran-2-yl)methyl
pivalate (105)
3
4
Yield 68%, Oil; 1H NMR (CDCl3, 400MHz) δ 6.22 (tt, 1H, >C=CH(CH2)16CH3), 4.25 (AB d, J =
11.8 Hz, 1H, (CH3)3(O)COCHH), 4.11 (AB d, J = 11.8 Hz, 1H, (CH3)3(O)COCHH), 3.63 (dq, J
= 6.76, 6.08Hz, 2H, HOCH2), 2.87 (AB q, J = 16.68 Hz, 1H, H-3a), 2.66-2.71 ( m, 3H, H-
3b,>C=CHCH2-), 2.09 (t, 1H, HOCH2-), 1.23 (s, 30H, >C=CHCH2(CH2)15CH3), 1.17 (s, 9H,
(CH3) 3(O)COCH2), 0.85 (t, J = 7.04 Hz, 3H, Alkyl-CH3); MS (FAB) m/z 481 (M+H)
5
6
7
8
9
10
11
12
13
14
15
16
17
4.1.27. (E)-(2-(Hydroxymethyl)-4-octadecylidene-5-oxotetrahydrofuran-2-yl)methyl
pivalate (106)
1
Yield 62%, Oil; H NMR (CDCl3, 400MHz) δ 6.75 (tt, J = 3.12, 3.08, 2.96 Hz, 1H,
>C=CH(CH2)16CH3), 4.27 (AB d, J = 11.8 Hz, 1H, (CH3)3(O)COCHH), 4.12 (AB d, J = 11.8 Hz,
1H, (CH3)3(O)COCHH), 3.66 (dq, J = 7.04, 6.6Hz, 2H, HOCH2), 2.80 (AB q, J = 16.88 Hz, 1H,
H-3a), 2.63 (AB d, J = 16.88 Hz, 1H, H-3b), 2.14 (m, 3H, HOCH2-, >C=CHCH2(CH2)15CH3),
1.23 (s, 30H, >C=CH(CH2)15CH3), 1.17 (s, 9H, (CH3) 3(O)COCH2), 0.85 (t, J = 7.04 Hz, 3H, Al-
kyl-CH3); MS (FAB) m/z 481 (M+H)
18
19
20
21
22
23
24
25
26
Acknowledgements
This research was supported by research grants from the National Research Foundation of Korea
(NRF) (R11-2007-107-02001-0) and by the Intramural Research Program of the National Insti-
tutes of Health, Center for Cancer Research, NCI (Project Z1A BC 005270).
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